An essential step in the genetic engineering of cells to carry out a desired function, such as production of a desired protein, is to transform the cells with a DNA, which includes the genetic information necessary for the cells to carry out the function. However, before cells can be transformed with a DNA, they must be treated to be made competent for transformation.
Transformation of yeast cells presents special problems because such cells have cell walls, in addition to plasma membranes, as barriers to entry of DNA.
One common method for transforming yeast cells is to first form spheroplasts from the cells by enzymatically digesting away the cell walls of the cells, then treating the spheroplasts to transform them with the desired DNA, and finally treating the transformed spheroplasts to regenerate the whole cells, complete with cell walls. A typical transformation process, employing spheroplasts of cells of the methylotrophic yeast species Pichia pastoris, is described by Cregg et al., Mol. Cell. Biol. 5, 3376-3385 (1985).
Employing spheroplasts for transformation of yeast cells entails a number of disadvantages associated with the osmotic sensitivity of spheroplasts and the need to first make spheroplasts and then reform whole cells from them. In recognition of these problems, efforts have been made in the art to devise methods for transforming whole yeast cells.
Thus, Ito et al., J. Bacteriol. 153, 163-168 (1983), have reported a method for making competent for transformation and transforming whole cells of Saccharomyces cerevisiae. The method of Ito et al. involves incubating whole cells of Saccharmoyces cerevisiae in an aqueous solution, buffered at a pH of about 8, of a salt of an alkali metal, to render the cells competent for transformation; incubating the competent, whole cells in a solution, again buffered to a pH of about 8, with the DNA with which the cells are to be transformed and a salt of an alkali metal; adding to the DNA solution bathing the cells polyethylene glycol, with an average molecular weight of about 4000, to bring the polyethylene glycol concentration to about 35% (w/v); incubating the cells in the resulting solution; and heat shocking the cells by suspending the solution in a water bath at 42.degree. C. for five minutes and then returning the solution to room temperature. Among the salts of alkali metals tested by Ito et al., at 0.1 M concentration in both the solution for making the cells competent for transformation and the transforming solution (prior to addition of polyethylene glycol), lithium chloride resulted in four to eight times more transformants per microgram of transforming DNA than sodium chloride. Further, among the lithium salts tested lithium chloride yielded a significantly lower number of transformants per microgram of transforming DNA than lithium acetate, lithium nitrate and lithium sulfate, all of which yielded about the same number of transformants. Ito et al. reported that circular plasmid DNA transformed alkali metal salt-treated whole cells more efficiently than linearized plasmid DNA and that cells from mid- to late-log phase cultures, when treated with an alkali metal salt, were transformed more efficiently than cells from late-log to stationary phase cultures. Ito et al. reported maximum transformation efficiency when the ratio of the concentration of cells treated with the salt of an alkali metal to the concentration of the alkali metal salt was between about 10.sup.11 to about 10.sup.12 cells per mole.
Davidow et al., Curr. Genet. 10, 39-48 (1985), reported a method for making competent for transformation and transforming whole cells of the yeast Yarrowia lipolytica. The method of Davidow et al. involves incubating whole cells of Yarrowia lipolytica at about 10.sup.8 cells per ml in an aqueous solution, buffered at a pH of about 7.5 of 0.1 M lithium acetate to render the cells competent for transformation; incubating the competent, whole cells in a solution again buffered to a pH of about 7.5, of the DNA with which the cells are to be transformed, heterologous carrier DNA (E. coli DNA) HaeIII digested to a size range of less than 1 kilobase pair, and 0.1 M lithium acetate; adding to the DNA solution bathing the cells a solution of 0.1 M lithium acetate buffered to a pH of about 7.5 and containing about 40% (w/v) polyethylene glycol, with an average molecular weight of about 4000, to bring the polyethylene glycol concentration to about 35% (w/v) in the DNA-containing solution; incubating the resulting solution; and subjecting the cells in the polyethylene glycol solution to a heat shock by suspending the solution at 37.degree. C. for several minutes. Contrary to Ito et al., Davidow et al. reported that circular plasmid DNA transformed lithium acetate-treated whole cells less efficiently than linearized plasmid DNA. Similar to Ito et al., Davidow et al found that cells from cultures in later stages of growth gave more transformants per microgram of transforming DNA than cells from cultures in early stages of growth. Davidow et al. reported that more than about 50 micrograms of the HaeIII digested carrier DNA per microgram of transforming DNA significantly increased the number of transformants obtained per microgram of transforming DNA; and sonicated E. coli DNA (size range 0.5-9 kbp) as carrier, present at 50 micrograms per microgram of transforming DNA, resulted in only half as many transformants per microgram of transforming DNA as HaeIII digested E. coli DNA (size range less than 1 kbp) as carrier also present at 50 micrograms per microgram of transforming DNA.
The differing results of Ito et al. and Davidow et al. illustrate the unpredictability of the effectiveness for any particular species of yeast of processes for making competent for transformation and transforming whole cells. In fact, whether methods found to be effective with one species of yeast will be effective with another species, on which the methods have not been tested, is highly uncertain. The properties of cell walls and associated membranes which affect competence for transformation and transformation efficiency remain obscure for all species and, particularly, species of yeast. It is thought that the chemical and physical properties of cell walls and membranes differ significantly among species of yeast, but how these differences might affect methods for making yeast competent for transformation or for transforming them, and the efficiency with which any particular method will function with a particular species, remain unknown.
The more distant taxonomically two species of yeast are, the less likely it is that observations concerning whole cell transformation methods made with one of the species will hold true for the other. In particular, nothing is known in the art about whole cell transformation methods applied to methylotrophic yeasts, such as those of the species Pichia pastoris. Methylotrophic yeasts differ significantly in numerous properties, including properties pertinent to processes for making cells competent for transformation and for transforming them, from non-methylotrophic yeasts such as S. cerevisiae and Y. lipolytica.